Gene deleted in colorectal cancer of humans

ABSTRACT

A new human gene termed DCC is disclosed. Methods and kits are provided for assessing mutations of the DCC gene in human tissues and body samples. Insertion, deletion, and point mutations in DCC are observed in human tumor cells. Normal tissues express DCC while most colorectal cancers do not. Loss of wild-type DCC genes is associated with neoplastic progression and a diminished life expectancy.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of grant numbersGM07309, GM07184, CA09243, and CA35494, awarded by the NationalInstitutes of Health.

This application is a continuation, of application Ser. No. 07/460,981,filed Jan. 4, 1990, now abandoned

TECHNICAL AREA OF THE INVENTION

The invention relates to the area of cancer diagnostics. Moreparticularly, the invention relates to detection of the loss and/oralteration of wild-type DCC genes in tumor tissues.

BACKGROUND OF THE INVENTION

Recent studies have elucidated several genetic alterations that occurduring the development of colorectal tumors, the most common of whichare deletions of the short arm of chromosome 17 (17p) and the long armof chromosome 18 (18q). Vogelstein, et al., Science, vol. 244, p. 207(1989); Fearon, et al., Science, vol. 238, p. 193 (1987); Muleris, etal., Ann. Genet. (Paris), vol. 28, p. 206 (1985); Monpezat, et al., Int.J. Cancer, vol. 41, p. 404 (1988). While some genetic alterations suchas RAS gene mutations appear to occur relatively early during coloretaltumor development, chromosome 18q deletions are often late eventsassociated with the transition from Class II to Class III adenomas orthe transition from the benign (adenomatous) to the malignant(carcinomatous) state, (Vogelstein et al., New England Journal ofMedicine, Vol. 319, p. 525, 1988) and appear to be related to metastasisand decreased survival time (Kern, et al., JAMA, vol. 261, pp. 13099-13103, 1989). Because carcinomas are often lethal, while the precursoradenomas are uniformly curable, the delineation of the molecular eventsmediating this transition are of considerable importance.

Allelic deletions have been reported to encompass a large area ofchromosome 18q. (Vogelstein, et al., ibid.) This area is known tocontain the BCL-2 gene (Tsujimoto, et al., Science, vol. 226, p. 1097(1984); and Cleary, et al., Cell, vol. 47, p. 19 (1986),) thegastrin-releasing peptide gene (Spindel, et al., Proc. Natl. Acad. Sci.,USA, vol. 81, p. 5699 (1984),) and the cellular homologue of the YES-1oncogene (Semba, et al., Science, vol. 227, p. 1038 (1985) and Yoshida,et al., Cytogenet. Cell Genet., vol. 40, p. 786 (1985)). All of thesegenes are known to be associated with cancers. If a particular region ofthe chromosome is the target of the deletions, i.e., it is involved inthe neoplastic process, precise delineation of the region is necessaryto provide methods of diagnosis as well as therapy.

According to the model of Knudson for tumorigenesis (Cancer Research,vol. 45, p. 1482, 1985), there are tumor suppressor genes in all normalcells which, when they become non-functional due to mutation, causeneoplastic development. Evidence for this model has been found in thecases of retinoblastoma and colorectal tumors. The implicated suppressorgenes in those tumors, RB and p53, were found to be deleted or alteredin many cases of the tumors studied. There is a need in the art ofcancer diagnosis and therapy to find other suppressor genes involved intumorigenesis, so that defects in the suppressor genes or effected cellscan be detected and the defects cured to abate or reverse the neoplasticprocesses.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method fordiagnosing and prognosing a neoplastic tissue of a human.

It is another object of the invention to provide a method of supplyingwild-type DCC gene function to a cell which has lost said gene function.

It is yet another object of the invention To provide a kit fordetermination of the nucleotide sequence of the DCC gene by thepolymerase chain reaction.

It is still another object of the invention to provide nucleic acidprobes for detection of mutations in the human DCC gene.

It is another object of the invention to provide a method of detectinggenetic predisposition to cancer.

It is still another object of the invention to provide a cDNA moleculeencoding the DCC gene product.

It is yet another object of the invention to provide a preparation ofthe human DCC protein.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment of thepresent invention a method of diagnosing or prognosing a neoplastictissue of a human is provided comprising: isolating a tissue from ahuman; and detecting loss of wild-type DCC genes or their expressionproducts from said tissue, said loss indicating neoplasia of the tissueand correlating with metastasis and early death.

In another embodiment of the present invention a method is provided forsupplying wild-type DCC gene function to a cell which has lost said genefunction by virtue of a mutation in the DCC gene, comprising:introducing a wild-type DCC gene into a cell which has lost said genefunction such that said wild-type gene is expressed in the cell.

In another embodiment a method of supplying wild-type DCC gene functionto a cell is provided comprising introducing a portion of a wild-typeDCC gene into a cell which has lost said gene function such that saidportion is expressed in the cell, said portion encoding a part of theDCC protein which is required for non-neoplastic growth of said cell.

In yet another embodiment a kit is provided for determination of thenucleotide sequence of the DCC gene by polymerase chain reaction. Thekit comprises: a set of pairs of single stranded DNA primers, thesequence of said set derived from chromosome 18q, said set allowingsynthesis of all nucleotides of the DCC gene coding sequences.

In still another embodiment of the invention a nucleic acid probe isprovided which is complementary to human wild-type DCC gene codingsequences and which can form mismatches with mutant DCC genes, therebyallowing their detection by enzymatic or chemical cleavage or by shiftsin electrophoretic mobility.

In another embodiment a particular nucleic acid probe is provided whichhybridizes to a DCC intron which is subject to insertional mutations intumor cells.

In still another embodiment of the invention a method is provided fordetecting the presence of a neoplastic tissue in a human. The methodscomprise isolating a body sample from a human; detecting in said sampleloss of a wild-type DCC gene sequence or wild-type DCC expressionproduct, said loss indicating the presence of a neoplastic tissue in thehuman.

In yet another embodiment a method is provided of detecting geneticpredisposition to cancer in a human, comprising: isolating a humansample selected from the group consisting of blood and fetal tissue;detecting loss of wild-type DCC gene coding sequences or theirexpression products from the sample, said loss indicating predispositionto cancer.

In still other embodiments a cDNA molecule is provided which comprisesthe coding sequence of the DCC gene.

In even another embodiment a preparation of the human DCC protein isprovided which is substantially free of other human protein The aminoacid sequence of the protein is shown in FIG. 5.

The present invention provides the art with the information that the DCCgene, a heretofore unknown gene is, in fact, the target of deletional,insertional, and point mutational alterations on chromosome 18q and thatthese alterations are associated with the process of tumorigenesis. Thisinformation allows highly specific assays to be performed to assess theneoplastic status of a particular tissue or the potential neoplasticstatus of an individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a map of the chromosomal walk and cross-hybridizingfragments in the DCC region. The DNA region of approximately 370 kb,cloned in 30 rounds of walking, is shown; only the maximal walk for eachof the rounds is shown. The map position at "O" marks the location ofp15 -65. An EcoRI map for the region was constructed and the EcoRIfragments that hybridized at reduced stringency (55° C.) to rodent,chicken, or Xenopus DNA samples are indicated by solid boxes andalphabetical letters (A-X). Human fragments G, I, J, K, M, O, and P wereused to isolate rat clones. The minimal region of cross-hybridizationwas identified and sequenced for both human and rat fragments. Thelocations of the EcoRI fragments that hybridized to human cDNA clonesare indicated by arrows.

FIG. 2 shows autoradiographs of Southern blots of DNA from mouse (M),rat (R), hamster (H), chicken (C), Xenopus laevis (F), and S. cerevisiae(Y) hybridized to human fragment O (in the panel on the left) and humanfragment P (in the panel on the right). The sizes of the correspondingmolecular weight markers in kilobases are indicated between the twopanels.

FIG. 3 shows the nucleotide sequence, predicted amino acid (AA) sequence(in single letter code) and splice acceptor and donor features of somehuman fragments and their corresponding rat homologues. Nucleotidesequences of the rat fragments were identical to those of the humanexcept where indicated. When the predicted amino acid sequence of therat differed from that of the human, the human sequence is given on theleft of the slash and the rat sequence on the right. The regions inhuman fragment O and P where no corresponding sequence was available forthe rat are indicated by the dashed lines. The predicted intron-exonboundaries are indicated by solid arrowheads, and the potential lariatsignal preceding the splice acceptor sequence is overlined.

FIG. 4 shows the nucleotide sequence derived from overlapping cDNAclones, prepared from mRNA of either the H82 cell line or from normalhuman brain. The methionine codon initiating the open reading frame(ORF) is designated as amino acid 202 and the last amino acid isnumbered 1648.

FIG. 5 demonstrates expression of DCC in human tumors and colorectaltumor cell lines. RNA was isolated and used as template to prepare afirst strand of cDNA. The cDNA samples were used for polymerase chainreaction (PCR) analysis. The PCR products were separated byelectrophoresis through an agarose gel, and after Southern transfer,hybridized to a radioactively-labeled subclone of fragment P. RNA wasisolated from normal human brain (lane 1), four different normal colonicmucosa specimens (lanes 2-5) or colorectal carcinoma cell lines (lane6-16).

FIG. 6A shows an autoradiograph of a Southern blot prepared from DNA ofperipheral blood lymphocytes of two normal individuals hybridized to aradiolabeled cDNA probe (pDCC 1.65). pDCC 1.65 contained nucleotides 591to 2250 of the cDNA shown in FIG. 4. The sizes of the fragments in kbare indicated to the right of the blot. A 0.45 kb EcoRI fragmentdetected by pDCC 1.65 migrated off the gel and is thus not in the FIG.

FIG. 6B shows a Southern blot using probe pKC 430 (from the 3'region ofthe 1.65 kb cDNA) to normal (N) and cryostat sections of tumor (T) DNAfrom S115. This probe contained nucleotides 1760 to 2205 of the cDNAshown in FIG. 5.

FIG. 7A shows Southern blots of DNA from normal and tumor DNA samplesdigested with EcoRI and Eco0109. The DNA was then hybridized to a 0.4 kbgenomic fragment which contained the exon from fragment P. Molecularweights in kb are indicated at the left; arrowheads indicate the tumorDNA fragments with insertions. DNA samples were derived from: lanes 1and 2: peripheral blood lymphocytes from two normal individuals; lanes 3and 4: non-neoplastic colon and colorectal carcinoma xenograft,respectively, from patient Cx7; lanes 5 and 6: non-neoplastic colon andcryostat sections of colorectal carcinoma, respectively, from patientS175; lane 7: carcinoma xenograft from patient Cx10;lane 8: colorectalcarcinoma cell line RKO; lane 9: colorectal tumor cell line Vaco6; lane10: colorectal carcinoma cell line SNU630; lane 11: colorectal carcinomacell line SW48; lane 12: colorectal carcinoma cell line HCT16.

FIG. 7B shows the sequence of the 170 bp XbaI-Eco0109 frag-ment to whichthe insertions shown in panel A were localized. The numbers above thesequence indicate the distance in bp from the 3' end of the exoncontained in fragment P. The XbaI and Eco0109 restriction sites areindicated. The two regions of TA repeats are overlined, and the 130 bpregion of alternating purine-pyrimidine sequence is contained betweenthe arrowheads.

FIG. 8A shows a comparison of the sequence homology of the fourimmunoglobulin-like domains of DCC with one another and with chickenN-CAM [N-CAM(c)] and mouse N-CAM [N-CAM(m)]. The N-CAM(c) and N-CAM(m)sequences shown represent the consensus of the five Ig-like domainspresent in each protein; if no consensus was present at a particularposition of N-CAM(c) or N-CAM(m) (i.e., if no two domains contained theidentical residue), then the position is indicated by an X. Spacesinserted for alignment are indicated by a dash. The conserved cysteinesthought to be involved in intra-domain disulfide pairing are indicatedby solid triangles; other amino acid residues highly conserved in N-CAMand other similar Ig-like domains of the C2 class (Williams, et al.,Ann. Rev. Immunol., vol. 6, p. 381, (1988)) are noted by open triangles.Sequences were aligned by inspection to give the greatest overall match.Residues in two or more of the DCC domains were boxed if they wereidentical. The N-CAM(c) and N-CAM(m) consensus sequences were boxed ifthey matched the DCC consensus.

FIG. 8B shows a comparison of the sequence homology between DCC andchicken and mouse N-CAM in the fibronectin-type III-related regions.

DETAILED DESCRIPTION

It is a discovery of the present invention that mutational eventsassociated with tumorigenesis occur in a previously unknown gene onchromosome 18q named here the DCC (Deleted in Colorectal Carcinomas)gene. Although it was previously known that deletion of alleles onchromosome 18q were common in certain types of cancers, it was not knownthat the target gene of these deletions was the DCC gene. Further it wasnot known that other types of mutational events in the DCC gene are alsoassociated with cancers. The mutations of the DCC gene involve grossrearrangements such as insertions and deletions. However pointmutations, which lead to loss of expression of wild-type DCC have alsobeen observed.

According to the diagnostic and prognostic method of the presentinvention, loss of the wild-type gene is detected. The loss may be dueto either insertional, deletional or point mutational events. If only asingle allele is mutated, an early neoplastic state is indicated.However, if both alleles are mutated then a late neoplastic state isindicated. The finding of DCC mutations thus provides both diagnosticand prognostic information. A DCC allele which is not deleted (e.g.,that on the sister chromosome to a chromosome carrying a DCC deletion)can be screened for other mutations, such as insertions, smalldeletions, and point mutations. It is believed that most mutations foundin tumor tissues will be those leading to greatly decreased expressionof the DCC gene product. However, mutations leading to non-functionalgene products would also lead to a cancerous state. Point mutationalevents may occur in regulatory regions, such as in the promoter of thegene, leading to loss or diminution of expression of the mRNA. Pointmutations may also abolish proper RNA processing, leading to loss ofexpression of the DCC gene product.

In order to detect the loss of the wild-type DCC gene in a tissue, it ishelpful to isolate the tissue free from surrounding normal tissues.Means for enriching a tissue preparation for tumor cells are known inthe art. For example, the tissue may be isolated from paraffin orcryostat sections. Cancer cells may also be separated from normal cellsby flow cytometry. These as well as other techniques for separatingtumor from normal cells are well known in the art. If the tumor tissueis highly contaminated with normal cells, detection of mutations is moredifficult.

Detection of point mutations may be accomplished by molecular cloning ofthe allele (or alleles) present in the tumor tissue and sequencing thatallele(s) using techniques well known in the art. Alternatively, thepolymerase chain reaction can be used to amplify gene sequences directlyfrom a genomic DNA preparation from the tumor tissue. The DNA sequenceof the amplified sequences can then be determined. The polymerase chainreaction itself is well known in the art. See, e.g., Saiki et al.,Science, Vol. 239, p. 487, 1988; U.S. Pat. No. 4,683,203; and U.S. Pat.No. 4,683,195. Specific primers which can be used in order to amplifythe gene will be discussed in more detail below. Insertions anddeletions of genes can also be detected by these techniques. Inaddition, restriction fragment length polymorphism (RFLP) probes for thegene or surrounding marker genes can be used to score loss of an alleleor an insertion in a polymorphic fragment. Other techniques fordetecting insertions and deletions as are known in the art can be used.

Loss of wild-type genes can also be detected on the basis of the loss ofa wild-type expression product of the gene. Such expression productsinclude both the mRNA as well as the protein product itself. Pointmutations may be detected by amplifying and sequencing the mRNA or viamolecular cloning of cDNA made from the mRNA. The sequence of the clonedcDNA can be determined using DNA sequencing techniques which are wellknown in the art. The cDNA can also be sequenced via the polymerasechain reaction (PCR) which will be discussed in more detail below.

Mismatches, according to the present invention are hybridized nucleicacid duplexes which are not 100% homologous. The lack of total homologymay be due to deletions, insertions, substitutions or frameshiftmutations. Mismatch detection can be used to detect point mutations inthe gene or its mRNA product. While these techniques are less sensitivethan sequencing, they are simpler to perform on a large number oftumors. An example of a mismatch cleavage technique is the RNaseprotection method, which is described in detail in Winter et al., Proc.Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers et al., Science,Vol. 230, p. 1242, 1985. In the practice of the present invention themethod involves the use of a labeled riboprobe which is complementary tothe human wild-type gene coding sequence. The riboprobe and either mRNAor DNA isolated from the tumor tissue are annealed (hybridized) togetherand subsequently digested with the enzyme RNase A which is able todetect some mismatches in a duplex RNA structure. If a mismatch isdetected by RNase A, it cleaves at the site of the mismatch. Thus, whenthe annealed RNA preparation is separated on an electrophoretic gelmatrix, if a mismatch has been detected and cleaved by RNase A, an RNAproduct will be seen which is smaller than the full-length-duplex RNAfor the ribeprobe and the mRNA or DNA. The riboprobe need not be thefull length of the DCC mRNA or gene but can be a segment of either. Ifthe riboprobe comprises only a segment of the DCC mRNA or gene it willbe desirable to use a number of these probes to screen the whole mRNAsequence for mismatehes.

In similar fashion, DNA probes can be used to detect mismatches throughenzymatie or chemical cleavage. See, e.g., Cotton et al., Proc. Natl.Acad. Sci. USA, vol. 85, 4397, 1988; and Shenk et al., Proc. Natl. Acad.Sci. USA, vol. 72, p. 989, 1975. Alternatively, mismatehes can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, HumanGenetics, vol. 42, p. 726, 1988. With either riboprobes or DNA probes,the cellular mRNA or DNA which might contain a mutation can be amplifiedusing PCR (see below) before hybridization. Changes in DNA of the DCCgene can also be detected using Southern hybridization, especially ifthe changes are gross rearrangements, such as deletions and insertions.

DNA sequences of the DCC gene from the tumor tissue which have beenamplified by use of polymerase chain reaction may also be screened usingallele-specific probes. These probes are nucleic acid oligomers, each ofwhich contains a region of the DCC gene sequence harboring a knownmutation. For example, one oligomer may be about 30 nucleotides inlength, corresponding to a portion of the DCC gene sequence. By use of abattery of such allele-specific probes, the PCR amplification productscan be screened to identify the presence of a previously identifiedmutation in the DCC gene. Hybridization of allele-specific probes withamplified DCC sequences can be performed, for example, on a nylonfilter. Hybridization to a particular probe under stringenthybridization conditions indicates the presence of the same mutation inthe tumor tissue as in the allele-specific probe.

Loss of DCC mRNA expression can be detected by any technique known inthe art. These include Northern analysis, PCR amplification and RNaseprotection. Diminished mRNA expression indicates a loss of the wild-typeDCC gene.

Loss of wild-type DCC genes can also be detected by screening for lossof wild-type DCC protein. For example, monoclonal antibodiesimmunoreactive with DCC can be used to screen a tissue. Lack of antigenwould indicate a DCC mutation. Antibodies specific for mutant allelescould also be used to detect mutant DCC gene product. Such immunologicalassays could be done in any convenient format known in the art. Theseinclude Western blots, immunohistochemical assays and ELISA assays. Anymeans for detecting an altered DCC protein can be used to detect loss ofwild-type DCC genes. Finding a mutant DCC gene product indicates loss ofa wild-type DCC gene.

Mutant DCC genes or gene products can also be detected in other humanbody samples, such as, serum, stool, urine and sputum. The sametechniques discussed above for detection of mutant DCC genes or geneproducts in tissues can be applied to other body samples. Cancer cellsare sloughed off from tumors and appear in such body samples. Inaddition, the DCC gene product itself may be secreted into theextracellular space and found in these body samples even in the absenceof cancer cells. By screening such body samples, a simple earlydiagnosis can be achieved for many types of cancers. In addition, theprogress of chemotherapy or radiotherapy can be monitored more easily bytesting such body samples for mutant DCC genes or gene products.

The methods of diagnosis of the present invention are applicable to anytumor in which DCC has a role in tumorigenesis. The diagnostic method ofthe present invention is useful for clinicians so that they can decideupon an appropriate course of treatment. For example, a tumor displayingloss of both DCC alleles might suggest a more aggressive therapeuticregimen than a tumor displaying loss of only one DCC allele.

The primer kit of the present invention is useful for determination ofthe nucleotide sequence of the DCC gene using the polymerase chainreaction. The kit comprises a set of pairs of single stranded DNAprimers which can be annealed to sequences within or surrounding the DCCgene on chromosome 18q in order to prime amplifying DNA synthesis of theDCC gene itself. The complete set allows synthesis of all of thenucleotides of the DCC gene coding sequences, i.e., the exons. The setof primers preferably allows synthesis of beth intron and exonsequences, as a number of DCC mutations have been found in a DCC intron.The kit can also contain DNA polymerase, preferably Taq polymerase, andsuitable reaction buffers. Such components are known in the art.

In order to facilitate subsequent cloning of amplified sequences,primers may have restriction enzyme sites appended to their 5 'ends.Thus, all nucleotides of the primers are derived from DCC sequences orsequences adjacent to DCC except the few nucleotides necessary to form arestriction enzyme site. Such enzymes and sites are well known in theart. The primers themselves can be synthesized using techniques whichare well known in the art. Generally, the primers can be made usingsynthesizing machines which are commercially available. Given thesequence of the DCC open reading frame shown in FIG. 4, design ofparticular primers is well within the skill of the art.

The nucleic acid probes provided by the present invention are useful fora number of purposes. They can be used in Southern hybridization togenomic DNA and in the RNase protection method for detecting pointmutations already discussed above. The probes can be used to detect PCRamplification products. They may also be used to detect mismatches withthe DCC gene or mRNA using other techniques. Mismatches can be detectedusing either enzymes (e.g., S1 nuclease), chemicals (e.g., hydroxylamineor osmium tetroxide and piperidine), or changes in electrophoreticmobility of mismatched hybrids as compared to totally matched hybrids.These techniques are known in the art. See, Cotton, supra, Shenk, supra,Myers, supra, Winter, supra, and Novaek et al., Proc. Natl. Acad. Sci.USA, vol. 83, p. 586, 1986. Generally, the probes are complementary toDCC gene coding sequences, although probes to certain introns are alsocontemplated. One probe in particular hybridizes to the XbaI-Eco0109fragment located 165 bp downstream from the DCC exon in fragment P shownin FIG. 3. Another probe hybridizes to fragment P itself, and is a probefor DCC ceding sequences. An entire battery of nucleic acid probes isused to compose a kit for detecting loss of wild-type DCC genes. The kitallows for hybridization to the entire DCC gene. The probes may overlapwith each other or be contiguous.

If a riboprobe is used to detect mismatches with mRNA, it iscomplementary to the mRNA of the human wild-type DCC gene. The riboprobethus is an anti-sense probe in that it does not cede for the DCC proteinbecause it is of the opposite polarity to the sense strand. Theriboprobe generally will be radioactively labeled which can beaccomplished by any means known in the art. If the riboprobe is used todetect mismatches with DNA it can be of either polarity, sense oranti-sense. Similarly, DNA probes also may be used to detect mismatches.

Nucleic acid probes may also be complementary to mutant alleles of DCCgene. These are useful to detect similar mutations in other patients onthe basis of hybridization rather than mismatches. These are discussedabove and referred to as allele-specific probes. As mentioned above, theDCC probes can also be used in Southern hybridizations to genomic DNA todetect gross chromosomal changes such as deletions and insertions. Theprobes can also be used to select cDNA clones of DCC genes from tumorand normal tissues. In addition, the probes can be used to detect DCCmRNA in tissues to determine if expression is diminished as a result ofloss of wild-type DCC genes. Provided with the DCC ceding sequence shownin FIG. 4, design of particular probes is well within the skill of theordinary artisan.

According to the present invention a method is also provided ofsupplying wild-type DCC function to a cell which carries mutant DCCalleles. The wild-type DCC gene or a part of the gene may be introducedinto the cell in a vector such that the gene remains extrachromosomal.In such a situation the gene will be expressed by the cell from theextrachromosomal location. If a gene portion is introduced and expressedin a cell carrying a mutant DCC allele, the gene portion should encode apart of the DCC protein which is required for non-neoplastic growth ofthe cell. More preferred is the situation where the wild-type DCC geneor a part of it is introduced into the mutant cell in such a way that itrecombines with the endogenous mutant DCC gene present in the cell. Suchrecombination requires a double recombination event which results in thecorrection of the DCC gene mutation. Vectors for introduction of genesboth for recombination and for extrachromosomal maintenance are known inthe art and any suitable vector may be used. Methods for introducing DNAinto cells such as electroporation, calcium phosphate co-precipitationand viral transduction are known in the art and the choice of method iswithin the competence of the routineer. Cells transformed with thewild-type DCC-gene can be used as model systems to study cancerremission and drug treatments which promote such remission.

Polypeptides which have DCC activity can be supplied to cells whichcarry mutant or missing DCC alleles. The sequence of the DCC protein isdisclosed in FIG. 4. Protein can be produced by expression of the cDNAsequence in bacteria, for example, using known expression vectors.Alternatively, DCC can be extracted from DCC-producing mammalian cellssuch as brain cells. In addition, the techniques of synthetic chemistrycan be employed to synthesize DCC protein. Any of such techniques canprovide the preparation of the present invention which comprises the DCCgene product having the sequence shown in FIG. 4. The preparation issubstantially free of other human proteins. This is most readilyaccomplished by synthesis in a microorganism or in vitro. Active DCCmolecules can be introduced into cells by microinjection or by use ofliposomes, for example. Alternatively, some such active molecules may betaken up by cells, actively or by diffusion. Extracellular applicationof DCC gene product may be sufficient to effect tumor growth, as DCC mayact at the cell surface like its homologues, the neural cell adhesionmolecules. Supply of molecules with DCC activity should lead to apartial reversal of the neoplastic state. Other molecules with DCCactivity may also be used to effect such a reversal.

The present invention also provides a preparation of antibodiesimmunoreactive with human DCC protein. The antibodies may be polyclonalor monoclonal and may be raised against native DCC protein or DCC fusionproteins. The antibodies should be immunoreactive with DCC epitopes,preferably epitopes not present on other human proteins. In a preferredembodiment of the invention the antibodies will immunoprecipitate DCCproteins from solution as well as react with DCC protein on Westernblots of polyacrylamide gels. Techniques for raising and purifyingantibodies are well known in the art and any such techniques may bechosen to achieve the preparation of the invention.

Predisposition to cancers can be ascertained by testing normal tissuesof humans for mutations of DCC gene. For example, a person who hasinherited a germline DCC mutation would be prone to develop cancers.This can be determined by testing DNA from any tissue of the person'sbody. Most simply, blood can be drawn and DNA extracted from the cellsof the blood. In addition, prenatal diagnosis can be accomplished bytesting fetal cells or amniotic fluid for mutations of the DCC gene.Loss of a wild-type DCC allele, whether for example, by point mutationor by deletion, can be detected by any of the means discussed above.

Molecules of cDNA according to the present invention are intron-free,DCC gene coding molecules. They can be made by reverse transcriptaseusing the DCC mRNA as a template. These molecules can be propagated invectors and cell lines as is known in the art. Such molecules have thesequence shown in FIG. 4. The cDNA can also be made using the techniquesof synthetic chemistry given the sequence disclosed herein.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention which has been described inbroad terms above.

EXAMPLE 1

This example demonstrates that the locus on chromosome 18q which is thesubject of frequent allelic deletions and of somatic mutations isexpressed in cell lines of lung and colorectal carcinoma.

Tumor S123 was found to have a different hybridization pattern fromnormal colonic mucosa of the same patient when probed with probe p15-65.The tumor displayed a heterozygous pattern with two MspI alleles of 7.8and 10.5 kb. The normal colonic mucosa was homozygous for the 7.8 kballele.

The p15-65 probe contains a 2.7 kb SalI-EcoRI fragment derived fromsequences adjacent to and including those contained within the anonymousDNA segment OLVII E10 Marlhens, et al., Nucl. Acids. Res., vol. 15, p.1348, 1987. OLVII E10 is an 0.8 kb Hind III-EcoRI fragment that marksthe D18S8 locus at 18q21.3 and detects MspI polymorphisms in normalindividuals. Plasmid p15-65 was derived from a human genomic DNA libraryprepared from human DNA partially digested with MboI and cloned inlambda FIX (Stratagene). Two phage clones hybridizing to OLVII E10 wereisolated by hybridization selection. Among several tested subfragmentsfrom the two phage clones identified, p15-65 produced the highest signalto noise ratio when used for Southern blot experiments. Plasmid p15-65(and OLVII E10) detect polymorphic MspI sites, and thus give rise tofour alleles of 10.5, 9.7, 7.8, and 7.0 kb at frequencies of 17%, 4%,49% and 30%, respectively (N=232).

In order to determine the molecular basis of the acquisition ofheterozygosity in the carcinoma tissue of patient S123, genomic DNAclones containing the affected MspI site were isolated from thecarcinoma and compared to normal DNA sequences. Except for MspI, therestriction maps to this region were identical in the tumor and normaltissue of patient S123 indicating no gross DNA additions or deletions inthe tumor. The affected MspI site was found to lie within an Alu-typerepeated element in a 1.8 kb EcoRI fragment which is 5 kb from p15-65.The sequence of the cloned DNA fragment from the S123 tumor DNA differedfrom the normal allele at a single base pair, resulting in thereplacement of the internal G residue within the MspI recognitionsequence 5'-CCGG-3' with an A residue. This mutation created a potential3'splice acceptor site, identical to the consensus sequence forintron-exon junctions of primate genes (Shapiro, et al., Nucleic AcidsRes., vol. 15, p. 7155, 1987; Ruskin, et al., Cell, vol. 38, p. 317,1984; and Zhuang, et al., Proc. Natl. Acad. Sci. USA, vol. 86, p. 2752,1989.) It is noted that mutations creating splice acceptor sitesassociated with abnormal RNA processing have been previously found ininherited diseases, such as thalassemias (Ley, et al., Proc. Natl. Acad.Sci., USA, vol. 79, p. 4775, 1982; and Sharp, Annual Reviews ofBiochem., vol. 55, p. 1119, 1986.)

Phage clones that encompassed a 35 kb region surrounding the mutatedMspI site of tumor S123 were isolated. All EcoRI fragments from thephage clones were subcloned and used in hybridization experiments withNorthern blots containing RNA of normal colonic mucosa and cell linesderived from tumors of the colon and several other organs. No expressionwas detected in these experiments, nor was expression detected in RNaseprotection studies of these RNA samples using selected subfragments fromthe phage clones. A more exhaustive strategy was therefore undertaken toidentify expressed from this region of chromosome 18q.

A bidirectional chromosomal walk from this region was carried out usingbacteriophage vectors. Over 140 unique clones, spanning approximately370 kb, were isolated in 30 rounds of walking; the clones representingthe maximal walk for each of the 30 rounds are shown in FIG. 1. Theclones were obtained as follows.

Human genomic DNA was partially-digested with MboI and fragments of12-18 kb were cloned in the lambda FIX vector (Stratagene) usingconditions recommended by the manufacturer. Clones were propagated in E.coli C600 or TAP 90 cells (Patterson, et al., Nucleic Acids Res., vol.15, p. 6298, 1987). For each round of walking, EcoRI maps wereconstructed from comparison of digests of overlapping phage clones(starting with page clones containing p15-65). EcoRI fragments mappingfurthest from previously obtained phage clones were used to re-screenthe library. Approximately 1 × 10⁶ phage clones were screened for eachround of walking; three to seven new clones were generally obtained ineach walk, and the clones purified through three rounds of hybridizationselection.

In an effort to identify potential exons in the 370 kb region on thebasis of their homology to other species, every EcoRI fragment from theregion was isolated and used as a hybridization probe at reducedstringency (conditions as described in Vogelstein, et al., Cancer Res.,vol. 47, p. 4806, 1987 except that the hybridization buffer contained0.5% non-fat dried milk and filter washing was performed in 44.5 mMsodium chloride, 1.8 mM sodium citrate, 0.3 mM Tris, pH7.5 at 55° C. for45 minutes) to screen DNA samples from various species (mouse, rat,hamster, chicken, Xenopus and yeast). Such a strategy for theidentification of exons was employed for the Duehenne muscular dystrophygene and cystic fibrosis genes. (Monaco, et al., Nature, vol. 323, p.646, 1986 and Rommens, et al., Science, vol. 245, p. 1059, 1989).Twenty-four of the 117 EcoRI fragments hybridized to discrete DNAfragments of at least one of the species tested; these fragments areindicated by the solid boxes in FIG. 1. The patterns observed with twoof the fragments producing strong cross-species hybridization areillustrated in FIG. 2. Fragment O hybridized strongly to mouse, rat,hamster, and Xenopus DNAs, and fragment P hybridized strongly to mouse,rat, and hamster DNAs.

Most of the cross-species hybridizing fragments were then used as probesto screen Northern blots prepared with RNA of various normal tissues ortumor cells lines, and also used to screen cDNA libraries from normalcolonic mucosa specimens, a colorectal adenoma cell line, a brain tumor,a fibrosarcoma line, and an embryonal carcinoma line. No evidence forexpression was obtained in these Northern blot studies, nor were anyhybridizing clones identified in any of the cDNA libraries using thecross-hybridizing fragments as probes.

In order to determine if any of the cross-hybridizing fragments hadexon-like structural features, we cloned several of the homologousfragments from the rat, and determined the regions ofcross-hybridization for each fragment for both human and rat.

Rat genomic clones were obtained in two ways. In some eases, fragmentsidentified on Southern blots by cross-species hybridization were elutedfrom agarose gels and cloned (as described for the human genomic clonesabove) using cross-hybridizing, radiolabeled human clones as probes. Inother eases, MboI partial digests of rat genomie DNA were cloned inlambda DASH (Stratagene) using conditions recommended by themanufacturer, and the library was screened with homologous human clonesof interest.

The cross-hybridizing regions were then subcloned and sequenced fromboth species; the seven regions for which this analysis was performedare G, I, J, K, M, O and P. The sizes of the regions of homology studiedranged from 128 to over 534 bp, and their homology ranged from 75-89%.The sequences were examined for open reading frames (ORFs), conservationin the predicted amino acid sequence of the ORFs, consensus mammaliansplice acceptor and lariat sequences at the 5' end of the ORFs, andconsensus splice donor sequences at the 3' region of the ORFs (Shapiro,et al., Nucleic Acids Res., vol. 15, p. 7155, 1987; and Ruskin et al.,Cell, vol. 38, p. 317, 1984). Several of these features were found inmost of the fragments sequenced. Three of the human-rat fragment pairs(fragments G, O, and P in FIGS. 1 and 3) were found to have moreexon-like features than the other fragments. The region of cross-specieshybridization from human fragment G and its homologous rat fragmentpredicted ORFs that differed at two amino acid positions (FIG. 3).Similarly, the ORFs predicted for the other two sets of fragments werehighly conserved with a single amino acid substitution distinguishinghuman fragments O and P from their respective rat homologues (FIG. 3).For all three sets of fragments, nucleotide substitutions werepredominantly at the third position of codons in the exon-like regions,and sequence homology decreased to 75% or less outside these regions.

The striking homology between the human and rat sequences and theirexon-like structural features suggested that the fragments might containpotential exons. However, as noted above, no expression was detectedwhen these three fragments were used as probes of Northern blots ofvarious RNAs or used to screen cDNA libraries. In addition, RNaseprotection experiments (Winter, Proc. Natl. Acad. Sci. USA, vol. 82, p.7575, 1985), using these three fragments to generate anti-sensetranscripts, failed to conclusively demonstrate evidence of expressionin a variety of RNA samples, including normal colonic mucosa, colorectalcell lines, and other tumor cell lines of various types.

To increase the sensitivity of expression assays, we utilized thepolymerase chain reaction (PCR, Saiki, et al., Science, vol. 239, p.487, 1988) in an "exon-connection" strategy. cDNA was prepared fromtotal RNA of various cell lines and tissues using reverse transcriptaseto prime synthesis with random hexamers (Noonan, et al., Nucleic AcidsRes., vol. 16, p. 10366, 1988). This single-stranded cDNA was then usedin PCR experiments with oligonucleotide pairs derived from the sequenceof two of the potential exons noted above. If the two potential exonswere present in the same RNA transcript, then, using the appropriateoligonucleotides, it would be possible to amplify a cDNA product linkingthese two regions. Trace amounts of DNA, which often contaminate RNApreparations, would not give rise to the same sized PCR product in thisassay because the exons were separated by an intron. Most pairs ofoligonucleotides derived from the seven regions of human-rat homologydescribed above did not generate discrete PCR fragments when tested bythis strategy. However, an oligonucleotide pair derived from thesequence of fragments O and P of FIG. 3 was found to generate a discrete233 bp PCR product from cDNA of several of the mRNA samples studies,including that derived from a small cell carcinoma of the lung (H82) anda colorectal carcinoma (HCT116).

The oligonucleotide derived from fragment O was5'-TTCCGCCATGGTTTTTAAATCA-3' and the oligonucleotide derived fromfragment P was 5'-AGCCTCATTTTCAGCCACACA-3'. Cycle times were 1 minute at95° C., 1 minute at 58° C., and 2 minutes at 70° C.; twenty-five cycleswere performed. The PCR products were phosphorylated using T4 DNA kinase(Bethesda Research Laboratories), ligated to EcoRI linkers (New EnglandBiolas), inserted into lambda gt10 phage arms (Stratagene), and clonedin E. coli C600 cells. Insert-containing clones were identified throughhybridization with radiolabeled probes from fragments O and P (FIG. 3).EcoRI inserts from the phage clones were subcloned in Bluescript SK(Stratagene) and sequenced as described by S. Tabor and C. C.Richardson, Proc. Natl. Atari. Sci. USA, vol. 84, p. 4767, 1987; andKraft, et al., Biotech., vol. 6, p. 544, 1988. The PCR products fromboth cell lines 482 and HCT 116 were found to be the product of splicingthe predicted exon of fragment O directly to the predicted exon offragment P. Thus the region of chromosome 18q is expressed.

EXAMPLE2

This example demonstrates the isolation and sequencing of the cDNAcorresponding to the coding sequence of the gene which is deleted incolorectal carcinomas.

To confirm and extend these PCR experiments, we constructed a cDNAlibrary from RNA of the H82 cell line (Gubler, et al., Gene, vol. 25, p.263, 1983). Approximately 3.0×10⁶ recombinant cDNA clones were screenedwith genomic DNA subclones containing regions of fragments G, O and P ofFIG. 3. Four hybridizing clones were isolated and mapped with respect toone another and to the genomic clones shown in FIG. 1. The longest clonewas 1.65 kb in length and hybridized to at least eleven unique EcoRIfragments in human genomic DNA (FIG. 6A), eight of which were presentwithin the 370 kb region cloned in the chromosomal walk shown in FIG. 1.The four cDNA clones isolated were sequenced and subsequently used asprobes of cDNA libraries obtained from H82 cells or from normal humanbrain to obtain additional cDNA clones extending for a total of 2854base pairs. Through sequencing, all clones were found to encodeoverlapping portions of a transcript in which there was a single longORF of 2250 bp, which extended to the end of the sequenced region. TheORF began with a methionine codon in a favorable context for translationinitiation (nucleotide I in FIG. 4) according to the paradigms of Kozak,Nucl. Acids Res., vol. 15, p. 8125, 1987. The methionine was followed bya relatively hydrophobic sequence of 25 amino acids which resembledpreviously described signal sequences associated with membrane-boundproteins (Watson, Nucleic Acids Res., vol. 12, p. 5145, 1984). Thesignal sequence was immediately followed by 725 amino acids withsignificant homology to the neural cell adhesion molecules and otherrelated cell surface glycoproteins. The gene encoding this transcriptwill be referred to as DCC (Deleted in Colorectal Carcinomas).

EXAMPLE 3

This example demonstrates that most normal tissues of rat and humanproduce the DCC transcript. However most colorectal tumor cell lines donot produce amounts as great as are produced in normal cells.

To identify the tissues in which the DCC gene was expressed, cDNA wasprepared from several rat organs. These were: liver, kidney, adrenal,heart, lung, stomach, esophagus, spleen, small bowel, breast, bladder,uterus, aorta, psoas, brain, colon, tongue and skin. Because of the highdegree of conservation of the DCC gene (see FIG. 3), the sameoligonueleotide primers used to demonstrate expression in human cellscould also be used in the rat. To assess expression, oligonueleotidepairs from fragments O and P of Figure a were used in a PCR expressionassay as described above.

Seventeen of the eighteen rat tissues tested appeared to produce thetranscript at low levels, with greatest abundance observed in brain(Chomczynski, et al., Anal. Biochem., vol. 162, p. 156, 1987). Only theliver did not produce detectable transcript. Similar analysis of humantissues and cell lines revealed that the transcript was present inhighest concentration in brain, and was also expressed in normal colonicmucosa and in several tumor cell lines, including those derived fromtumors of the lung, brain and mesenchyme (FIG. 5 and data not shown). Inmost colorectal carcinomas, however, expression was greatly reduced orabsent; of seventeen colorectal tumor cell lines studied, only twoexpressed DCC mRNA at levels in excess of 5% of that produced in normalcolonic mucosa (examples in FIG. 5). The human colorectal carcinoma celllines used were: lane 6, SW948; lane 7, SW1417; lane 8, SW1116; lane 9,SW403; lane 10, SW1463; lane 11, SW48; lane 12, HCT116; lane 13. RKO;lane 14, RCA; lane 15, "C"; lane 16, MOSER.

To determine the size of the transcript produced from this gene,Northern blots containing RNA from normal colonic mucosa or brain werehybridized with radioactively labelled cDNA clones. A major band of10-12 kb was observed in normal brain RNA, but no bands were seen in theRNA from colonic mucosa (data not shown), consistent with the higherlevel of expression observed in brain by PCR analysis.

EXAMPLE 4

This example demonstrates that the boundary of the deletion of one ofthe alleles in the S115 tumor is within the DCC gene and thatrearrangements of the gene can be detected in human cells using cDNAprobes.

In an attempt to establish the boundaries of the homozygous loss in theS115 tumor with respect to the DCC gene, the cDNA clones were used toprobe Southern blots containing S115 DNA. A 430 bp subclone (pKC430,representing nucleotide 1760 to 2205 of the cDNA) detected three EcoRIfragments of 20 kb, 10 kb, and 1.8 kb in DNA from non-neoplastic colonicmucosa of patient S115 (FIG. 6B). However, in DNA from the S115 tumor,the 20 kb fragment was not detected and the 10 kb and 1.8 kb fragmentswere present at approximately half the intensity observed in normal DNA.In addition, a new fragment of 5 kb was observed only in DNA from thetumor (marked with an arrowhead in FIG. 6B). Probes more 3'than pKC430also detected fragments in tumor DNA which were present at half theintensity of those seen in normal DNA, while probes 5'of pKC430 detectedfragments which were homozygously deleted in the tumor. Thus, the DCCgene appeared to be broken by the deletion event on one of the twocopies of chromosome 18, and the breakpoint established one boundary ofthe homozygous loss.

EXAMPLE 5

This example demonstrates that the cDNA probes of the present inventioncan be used to detect genetic alterations in DNA samples isolated fromcolorectal tumors.

To search for other genetic alterations, the cDNA probes were used inSouthern blot analysis of colorectal tumor DNA samples. DNA from normaland tumor DNA samples were digested with EcoRI and Eco0109 and Southernblots were prepared as described above. The DNA was then hybridized to a0.4 kb genomic fragment which contained the exon from fragment P.

Three of 51 primary rumors and two of twenty-one tumor xenografts werefound to have new fragments not present in normal DNA of the samepatient. In addition, five of twenty-two colorectal tumor cell lineswere found to have altered fragments not present in forty-four DNAsamples from normal individuals nor in any of the 45 DNA samples fromtumor cell lines derived from tissues other than that of the colon orrectum.

In all cases, detailed mapping experiments showed that the new fragmentsdetected by the cDNA probe resulted from insertions in an approximately170 kb XbaI-Eco0109 fragment located 165 bp downstream of the exon infragment P of FIG. 8. The insertions were mapped by comparison ofSouthern blot patterns produced by digestion of tumor DNA samples with acombination of XbaI and Eco0109, and HindIII. The size of the insertionin the tumors varied from 120-300 bp (FIG. 7A). Some variation in thesize of the Xbal-Eco0109 fragment in alleles from normal individuals wasseen; however, the maximal difference between the size of the smallestand largest of the 88 normal alleles studied was approximately 35 bp,and the largest of the normal alleles was found to be approximately 120bp smaller than any of the altered alleles seen in the tumors.

The 1.4 kb EcoRI fragment (fragment P) containing the insertion site wasisolated from one of the genomic clones of the chromosome walk (FIG. 1)and sequenced; the sequence of the 170 bp XbaI-Eco0109 fragment fromthis fragment is shown in FIG. 7B. There were two regions of TA repeatsin the XbaI-Eco0109 fragment; one of the regions had eight repeats andthe other had twenty-six. Both TA repeat regions were contained within a130 bp region of alternating purine-pyrimidine base pairs which couldpotentially form Z-DNA (Rich, et al., Annu. Rev. Biochem., vol. 53, p.791, 1984).

EXAMPLE 6

This example demonstrates the sequence similarity between fourimmunoglobulin-like domains of the DCC gene with the chicken N-CAM andmouse N-CAM genes.

The predicted amino acids sequence of DCC is highly homologous to theneural cell adhesion molecules (N-CAM) and other related cell surfaceglycoproteins. (Edelman, Biochem., vol. 27, p. 3535, 1988). Two areas ofhigh homology are noted. First the DCC gene contains fourimmunoglobulin-like (Ig-like) domains of the C2 class, defined by pairsof cysteines separated by 50 to 56 amino acids and other highlyconserved residues surrounding the first and second cysteine of eachpair. (See FIG. 8A and Williams, Ann. Rev. Immunol., vol. 6, p. 381,1988).

Sequences of the domains were aligned by inspection and spaces,indicated by dashes were inserted to give the greatest overall match.Residues in two or more of the DCC domains were boxed if they wereidentical. Each Ig-like domain is approximately 100 amino acids inlength. DCC domain no. 1 includes amino acids 40 to 139, domain no. 2includes amino acids 140-239, domain no. 3 includes amino acids240-332,and domain no. 4 includes amino acids 333-422. The four Ig-likedomains of DCC were found to be more homologous to one another than toN-CAM L1, or other members of the Ig superfamily. A consensus sequencefor the four Ig-like domains of DCC could be derived for 67% of thepositions; the DCC consensus sequence matched the N-CAM consensussequence at 42% of these positions.

Sequence homology between the DCC and chicken and mouse N-CAM genes wasalso found in the fibronectin-type III-related regions. DCC amino acidpositions 423 to 605 were compared to amino acids 481 to 662 of the twoN-CAM proteins. Potential sites of N-linked glycosylation are found atseveral positions within this region.

The fibronectin-type III-related domain is similar to thefibronectin-like domains present in N-CAM, L1, leucocyte common antigenrelated gene 1 (Larl), fasciclin II, and other members of the celladhesion molecule family. These fibronectin-related domains are carboxylto the Ig-like domain in all of these proteins including DCC. Of 195positions within the fibronectin-like region of DCC, 31% were identicalin DCC and N-CAM and several conservative substitutions were also found(FIG. 8B). The extensive homologies in both Ig-like and fibronectin-likedomains between DCC and other members of this family suggest that theseproteins may all have been derived from a common precursor that includedboth these regions.

EXAMPLE 7

This example demonstrates the expression of the DCC protein in bacteriaand the production of anti-DCC antibodies.

The DCC cDNA sequence shown in FIG. 4 was inserted in a bacterialexpression vector, pEX2. The vector will produce the protein of any openreading frame as a fusion product with betagalactosidase. (See Stanley,et al., The EMBO Journal, vol. 3, pp. 1429-1434, 1984.) The bacteriallyexpressed DCC protein was partially purified and injected into rabbitstogether with Freund's adjuvant. The rabbits were given three monthlybooster injections. The rabbits produced antibodies whichimmunoprecipitate DCC proteins synthesized in in vitro translationreactions. The antibodies also bind to DCC on Western blots.

I claim:
 1. An isolated, purified DNA molecule which encodes the aminoacid sequence shown in FIG. 4, amino acids 202 through
 1648. 2. The DNAmolecule of claim 1, wherein the sequence of said molecule consists of anucleotide sequence shown in FIG.
 4. 3. A vector comprising the DNAmolecule of claim
 1. 4. An isolated, purified DNA molecule comprisingthe coding sequence of the DCC gene, as shown in FIG. 4, nucleotides 605through
 4945. 5. A vector comprising the DNA molecule of claim
 4. 6. Ahost cell which replicates the cDNA sequence as shown in FIG. 4nucleotides 605 to
 4945. 7. The host cell of claim 6 which expresses theamino acid sequence shown in FIG. 4, amino acids 202 to 1648.